The overall objective of this pilot project is to investigate the role of androgens during adipogenic differentiation using in-vitro as well as in vivo models of adipogenesis, and understand the possible mechanisms by which androgens may regulate the fat cell formation. Android obesity, an important risk factor for cardiovascular diseases occurring at disproportionately high rates in racial/ethnic minorities, is characterized by excessive accumulation of adipose tissue in abdominal and visceral regions. Clinical studies using Testosterone (T) has suggested its beneficial role in men, with a significant increase in fat-free mass. However, the mechanisms by which T or its metabolite dihydrotestosterone (DHT) inhibit fat cell formation is largely unknown. In this project, we will study the adipogenic differentiation of pluripotent and preadipocyte cell lines and the possible roll of androgens in modulating the common differentiation pathway of these cells into adipogenic lineage. We will test the presence of androgen receptors in our cell system and focus our studies on the effects of androgen on the key transcription factors C/EBPalpha and PPARgamma2, which are necessary and sufficient to induce adipogenesis. We will examine the mRNA and protein expression of these factors using RT-PCR, western blot and immunohistochemistry. The fat cell formation will be quantitated using Oil-Red O staining. The possible intermediate role of Wnt signaling pathway, a molecular switch that governs adipogenesis, will be investigated during androgen mediated inhibition of adipogenesis. We will test whether inhibition of Wnt signaling using pharmacological as well as retroviral approaches blocks the anti-adipogenic potential of androgens. We will generate an in vivo model of adipogenesis by injecting 3T3-F442 preadipocyte cell line in athymic mice and study the effect of different doses of T and DHT on the formation of fat pads. The expression of adipose specific mRNAs and proteins like C/EBPalpha, PPARgamma2 and 422/aP2 will be quantitated and analyzed to examine the anti-adipogenic role of androgens.